Comprehensive evaluation of reversed-phase and hydrophilic interaction liquid chromatography - mass spectrometry for...

Gangliosides are biologically significant molecules with essential physiological roles in the organism. They are increasingly studied as biomarkers for disease diagnosis and progression. Due to their extreme structural diversity and typically low abundance in biological samples, efficient and sensitive liquid chromatography-mass spectrometry (LC-MS) methods are required for their analysis. In this study, we present a comprehensive comparison of the performance of two widely used LC-MS approaches: reversed-phase (RP) and hydrophilic interaction liquid chromatography (HILIC). Both methods were independently optimized following an identical protocol, with a focus on chromatographic performance and MS response. Notably, salt concentration had a pronounced impact on ganglioside retention, peak shape, isomer separation, and MS signal intensity in both chromatographic modes. In HILIC, adjusting the initial gradient composition and profile enabled effective separation of gangliosides from highly abundant matrix lipids. The RP method demonstrated superior ganglioside signal intensity and a 3.5-fold reduction in analysis time. Both approaches allowed the separation of gangliosides based on the number of sialic acid and saccharide residues. Additionally, the RP method enabled separation according to ceramide composition. We established rules for RP separation based on the number of carbon atoms in the ceramide moiety, which can be used to predict ganglioside retention times. Based on our findings, each method offers distinct advantages for specific analytical goals. The HILIC approach is better suited for untargeted ganglioside profiling using accurate m/z and interclass separation, while the RP method is ideal for sensitive, high-throughput targeted analysis in complex biological matrices.